An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

Abstract

Background: Xylose is a second most abundant sugar component of lignocellulose besides glucose. Efficient fermentation of xylose is important for the economics of biomass-based biorefineries. However, sugar mixtures are sequentially consumed in xylose co-fermentation with glucose due to carbon catabolite repression (CCR) in microorganisms. As xylose transmembrance transport is one of the steps repressed by CCR, it is therefore of interest to develop a transporter that is less sensitive to the glucose inhibition or CCR.

Results: The glucose facilitator protein Glf transporter from Zymomonas mobilis, also an efficient transporter for xylose, was chosen as the target transporter for engineering to eliminate glucose inhibition on xylose uptake. The evolution of Glf transporter was carried out with a mixture of glucose and xylose in E. coli. Error-prone PCR and random deletion were employed respectively in two rounds of evolution. Aided by a high-throughput screening assay using xylose analog p-nitrophenyl-beta-D-xylopyranoside (pNPX) in 96-well plates, a best mutant 2-RD5 was obtained that contains several mutations, and a deletion of 134 residues (about 28% of total residues), or three fewer transmembrane sections (TMSs). It showed a 10.8-fold improvement in terms of pNPX transport activity in the presence of glucose. The fermentation performance results showed that this mutant improved xylose consumption by 42% with M9 minimal medium containing 20 g L-1 xylose only, while with the mixture sugar of xylose and glucose, 28% more glucose was consumed, but no obvious co-utilization of xylose was observed. Further glucose fed-batch experiments suggested that the intracellular metabolism of xylose was repressed by glucose.

Conclusions: Through random mutagenesis and partial deletion coupled with high-throughput screening, a mutant of the Glf transporter (2-RD5) was obtained that relieved the inhibition of xylose transport by glucose. The fermentation tests revealed that 2-RD5 was advantageous in xylose and glucose uptakes, while no obvious advantage was seen for xylose co-consumption when co-fermented with glucose. Further efforts could focus on reducing CCR-mediated repression of intracellular metabolism of xylose. Glf should also serve as a useful model to further exploit the molecular mechanism of xylose transport and the CCR-mediated inhibition.

Figure 1

Secondary structure models of the wild type Glf and the mutant 2-RD5 predicated by HMMTOP. (A) Wild type Glf has 12 TMSs and both N- and C- termini are inside the cytoplasm. (B) Mutant 2-RD5 has 9 TMSs and N-terminus is on the outside of the cytoplasm, while C-terminus is on the inside of the cytoplasm.

Figure 2

Schematic overview of random deletion used in this study. A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and Klenow fragment. D) Single digested with Bgl II. E) Random ligation with T4 DNA ligase.

Figure 3

Xylose inhibition assay for Glf mutant 2-RD5. (A) Xylose inhibition of pNPX transport (normalized by dry cell weight) for BL21(DE3)/pET30a-glf(2-RD5)-xynB (gray columns) and BL21(DE3)/pET30a-glf-xynB (black columns). The inhibition assay was done in the presence of 2% glucose. (B) Relationships between the invert of the initial velocity of pNPX transport and the concentrations of xylose for BL21(DE3)/pET30a-glf(2-RD5)-xynB. All data represent triplicate measurements of same cultures.

Figure 4

Growth curves of E. coli BL21(DE3) cells containing BL21(DE3)/pET30a-glf(2-RD5)-xynB, BL21(DE3)/pET30a-glf-xynB and BL21(DE3)/pET30a. (A) in M9 minimal medium supplemented with 20 g L^-1 xylose. (B) in M9 minimal medium supplemented with 10 g L^-1glucose and 10 g L^-1 xylose. (C) in M9 minimal medium supplemented with 20 g L^-1 xylose, adding 5 g L^-1 glucose after 1 h IPTG induction. E. coli BL21(DE3)/pET30a (filled black square), wild type E. coli BL21(DE3)/pET30a-glf-xynB (filled black circle) and E. coli BL21(DE3)/pET30a-glf(2-RD5)-xynB (filled black triangle).

Figure 5

Sugar consumption in flask fermentation. (A) in minimal medium supplemented with 20 g L^-1 xylose, B) in minimal medium supplemented with 10 g L^-1 glucose and 10 g L^-1 xylose, C) in M9 minimal medium supplemented with 20 g L^-1 xylose, adding 5 g L^-1 glucose after 1 h IPTG induction. Sugars for different strains are: xylose of E. coli BL21(DE3)/pET30a (filled black square), xylose of wild type E. coli BL21(DE3)/pET30a-glf-xynB (filled black circle), xylose of E. coli BL21(DE3)/pET30a-glf(2-RD5)-xynB (filled black triangle), glucose of E. coli BL21(DE3)/pET30a (empty square), glucose of wild type E. coli BL21(DE3)/pET30a-glf-xynB (empty circle) and glucose of E. coli BL21(DE3)/pET30a-glf(2-RD5)-xynB (empty triangle).